GryphonCAST Episode 9 Transcript

Speakers: Michael Lim and Sydney Pascetta

A listener’s note, Sydney and I are not medical professionals so we recommend that you do not take our conversation as medical advice and instead talk to your healthcare provider. With that being said, on with the show.

Michael Lim
When you hear about challenging environments, you likely at first think of areas of overwhelming heat or sheer cold, like the deserts or Arctic tundra. However, special environments can be found within our very bodies, such as the creation of low oxygen or hypoxic microenvironments where tumors develop. As cancer cells multiply, they consume oxygen at a rate faster than our body can locally supply it. A new study suggests hypoxic environments increase the number and migration of cancer cells by increasing receptors for a specific neuro hormone called neuropeptide Y (NPY). Open your ears and mind and let's chat about that. Welcome to GryphonCAST, a podcast where we casually chat about science coming out of the College of Biological Science at the University of Guelph, and how their work can affect lives around the world. I'm your host, Michael Lim. With me today is special guest and PhD student Sydney Pascetta. And we'll be chatting about her recent work on neuropeptide Y or NPY and through receptors and breast tumors and this work can help with treating cancerous cells. Welcome Sydney. To orient yourself to our guests and I guess how to get you more comfortable, how would you describe your research and or the work done in the Uniacke Lab in general?

Sydney Pascetta 
So, our research focusses on low oxygen environments and how they affect cancer progression. So, if you imagine a tumor, a solid tumor, it actually does a really bad job at recruiting new vasculature. So, as you progress into the center of the tumor, those cells become more and more hypoxic or have less and less oxygen available to them. And traditionally, these would maybe be thought of as dead or non-functional cells, but they're typically the most aggressive cells in the tumor microenvironment. So, we're trying to understand what makes these cells unique in that environment. And maybe we could use more targeted therapies towards those cells and not something as like generally harmful as like chemo and stuff like that.

Michael Lim
It is a good point. When you think of hypoxic cells and cells needing oxygen to develop and grow, you expect that, you know, there's no oxygen they shouldn't be able to multiply so quickly.

Sydney Pascetta 
Yeah, but they're actually very aggressive. They're very metastatic. They have a lot of like migratory and proliferative, like capabilities. So that's interesting because it's unique, and hopefully it's like not found in the rest of your body. I mean, that's your body sits at what we term physioxia. So normal oxygen in the air, ambient oxygen is 21%. But most of your tissues are significantly lower than that and the range is usually from like 2 to 8%. Depending on the tissues, depending on how far away it is from your blood vessels, all that kind of stuff. Obviously, the lungs are like the most oxygenated, but your brain is like actually really low. So, it just depends on the area of the body.

Michael Lim 
So, why did you decide to go into this research? Was there a particular moment you're like, “Oh, yes, I'm going to study cancer cells and hypoxia”.

Sydney Pascetta  
No, actually, I really just like Jim (Uniacke) as an advisor. I went to Queen’s for my undergrad. And then I was looking at a few different universities as again, like U of T and Guelph, and I met with Jim and I really liked him. My research in my undergrad is on plant immune systems, so completely different, but and I also worked on like, gut polysaccharides for a summer too. So, I've just done a bunch of different things and I just really liked the lab environment more so than anything.

Michael Lim
It’s good to try a bunch of different things. Otherwise, how would you know what you want to do? [Sydney: Yeah, exactly]. So, speaking of research, you recently published a study titled “Expression of hypoxia inducible factor-dependent neuropeptide Y receptors Y1 and Y5 sensitizes hypoxic cells to NPY stimulation”. So, to help kind of guide our audience through that, can you describe what neuropeptide Y does in the body? And what's so special about Y1 and Y5 receptors specifically?

Sydney Pascetta 
Well neuropeptide Y is like the most abundant neuropeptide in the central nervous system. So, it has like a bunch of different functions, one of which the most classically explored function is actually feeding behavior and feeding regulation and stuff like that. But it also acts in vasoconstriction and cellular proliferation, and a bunch of other like developmental processes. So, the reason why we're actually looking at Y1 and Y5 specifically is because they're the most ubiquitous expressed amongst breast cancer cells specifically. So, there's a whole family there's six of them, one of which is nonfunctional in humans, and then the other ones just aren't really highly expressed in breast cancer.

Michael Lim
So, the most promising targets in other words. [Sydney: Yeah, exactly]. So as part of your research, just to clarify for people, we didn't just get a whole bunch of cancer patients to work with breast cancer cell lines. So MCF-7 and mda-mb-231. What's the difference between these two types of cell lines? And what are some benefits of working with cell lines versus you know, grabbing a random cancer patient from the hospital and saying; “Hi, can you be part of my study?”

Sydney Pascetta
Well actually, cell lines are derived from humans. So, they all originated from one donor human at some point in time, but they are immortalized cell lines. So, we're able to work with them in the lab continuously by doing something that we call “passaging them” so we're able to keep them alive. The difference between the two cell lines that we chose is that the MCF-7s are ER PR positive. So estrogen receptor, progesterone receptor positive, and mda’s are triple negative. If you've heard that kind of buzzword for breast cancer, triple negative breast cancer is typically viewed as like a very aggressive metastatic form of breast cancer. So, we chose two different cell lines in order to understand if the phenomena that we're observing is specific to one kind of like cellular microenvironment or if it can be kind of generalized across different breast cancers cell lines.

Michael Lim
So, the different cell lines used your study responded to hypoxia and DMOG (Dimethyloxalylglycine), which is a chemical used to mimic hypoxia at different timescales. So, you mentioned briefly that these are coming from different individuals with different types of breast cancer like the triple negative, for example. So, what do you think caused the differences in time between these two different cell lines?

Sydney Pascetta
Honestly, that's really hard to say. The cell lines themselves are quite different and like I said, the triple negative ones, the mda’s that we were talking about are typically viewed to be more metastatic and aggressive and have like a completely different phenotype than the MCF-7’s. They're quite different cell lines in general. So, they have different signalling pathways that are activated within each of them, generally speaking. So the difference is not surprising and you would expect there to be differences amongst them.

Michael Lim
Stimulations of the receptors Y1 and Y5 generally increase mitogen-activated protein kinase or MAPK signalling in the different cell lines when exposed to hypoxic conditions. So, considering the role of signalling in gene transcription and translation within cells, for our more lay audience, using your DNA to make necessary proteins that function in the body, what is this increased signalling mean for cancerous cells?

Sydney Pascetta
So, in that case, signalling is a really classically explored signalling pathway that a lot of different people look at for a lot of different reasons. But it's primary role is important for developmental processes. So, it's often activated like during embryogenesis, and growth, and development and all of those really important processes. But like the, I guess you could say the catch-22 about cancer is that cancer is typically a disease of normal developmental processes gone wrong. So, they're like kind of put into overdrive. So, proliferation, migration, all of these activities, cellular activities are actually normal and good for regular developmental processes but when these processes become hijacked. And they're happening at a much more frequent rate than they should be or maybe there's checkpoint proteins that aren't functioning properly so they can't kind of rein in these processes. That's when cancer kind of develops. So, by looking at this pathway, we're basically saying, “Can we inhibit or accelerate these different downstream effects?” So, the MATK assay that we did at the beginning was can we actually affect directly this pathway? And then what is the output of that? So that was like the proliferation and migration assays that were done later on in the paper.

Michael Lim
Oh, that makes sense. It kind of leads on to like, the next question. And that obviously, they mentioned before NPY are independent, why? And obviously receptors in that case, signaling is very important in terms of regular functions in the body and within regular cells. So, if cancer treatments reduce the ability for neuropeptide Y receptors to normally function because you're trying to suppress this overactivity. What does this mean for non-cancer cells within the body?

Sydney Pascetta
So, we wouldn't really look at it from that type of perspective. It would be more in terms of like a specialized therapy instead of chemotherapeutic. So, there's a whole new field of personalized kind of medicine and therapy for cancer that's exploring different ways that we can do pointed drug delivery instead of irradiating someone with like a chemotherapeutic, or radiation therapy and stuff like that. So, there are some labs out there that are looking at bioreductive prodrugs. So basically, when they get into an environment with a different oxygen concentration like hypoxia, that drug would become active or using nanoparticles for delivery or direct like intratumoral injections. So, we would be looking more at that like specified drug delivery format rather than affecting like the entire body.

Michael Lim
I was about to ask you like how do you keep it localized in the body because you know, transport within the body depending on what type of drug it is can be passed easily between cells. Like it's kind of more of a trying to control for hypoxia and take advantage of the hypoxic nature of cancers. 

Sydney Pascetta
Yeah, that's what I'd be hoping to do. And that's kind of like the ethos of our lab in general. So, like this project is one branch our lab, but our lab actually looks at very, very distinct projects. So there's one that looks at alternative splicing within the ribosome. There's another one that looks at a process called ISGylation which is kind of more immune system driven in a lot of cases. So yeah, there's like a lot of projects in our lab, but it's mostly understanding the biophysiology of the hypoxic microenvironment. And like I was saying before, kind of what is unique to that, and how we can maybe target that.

Michael Lim
So, we kind of mentioned this briefly before in that you're focusing on Y1 and Y5 five because they're highly prevalent within breast cancer cells. And so, you focus on those receptors as well as neuropeptide Y itself and look at possible treatments. So how applicable would you say your research is to towards studying other cancers and trying to address the receptors there by focusing on Y1 and Y5?

Sydney Pascetta
Yeah, so it would just depend on the expression levels of Y1 and Y5 in those other other types of cancer because they would all be unique and there are other cell lines that do express Y1 and Y5 to varying degrees. It just depends on the context and the person, really. But anything that's highly innervated by the central nervous system will have neuropeptide Y stimulation going on, but just like the proportion of expression of these two receptors would be in contrast with the other ones, etc.

Michael Lim
My brain is just kind of casting back from our earlier conversation of how it's different level between, you know, a hypoxic environment and like physiologically relevant hypoxic environment. So, when you're raising your cell lines, does that mean that a normal environment is actually hypoxic compared to a regular environment already? Sorry if that's a little confusing, but you know what I mean?

Sydney Pascetta
That’s actually a really, really important question in science and there's a whole project in our lab that focuses on physioxia, that range that I was talking about, that's actually biologically significant within your tissues. So most, most cell research that's done with cell lines raises those cell lines in incubators at 37 degrees, which is what the internal temperature of your body is, but they don't account for the ambient gases. So, in an incubator, those cells are raised at 21% because that's what ambient oxygen levels are. So actually, what another project in our lab has found is that that 21% oxygen range is actually quite stressful for cells. It's not their normal, physiologic, relevant environment. So, it's actually pretty stressful for them and we have another incubator where we can control what the oxygen concentration is. And in those more biologically relevant oxygen concentrations, cells experience less stress in general, like there's less DNA damage, and other processes going on. And then the reason why we choose hypoxia in this study is because again, it's relevant to the tumor microenvironment. So a hypoxic tumor is basically a tumor that's around 1% oxygen or less. 

Michael Lim 
So, it's all relative to a regular person. You're like, “Oh, yes, that's totally normal at 21%”. But to your body’s cells, it's a hyperoxic environment. You're causing excessive oxidation, causing DNA damage. Obviously, something you want to avoid. 

Sydney Pascetta 
Yeah, exactly and for most research is done at 21% so the way that we understand basic cellular function is for the most part done at 21%. So our lab is kind of bringing research to the table that shows that maybe this isn't, you know, the most relevant concentrations to raise them at in general and like, maybe I want to say that everything we understand about how cells function is at least slightly different from traditional view of it but it could be, you know.

Michael Lim
Do you think there ever been a moment where you're 100% sure of the connection between neuropeptide Y receptors and cancer, you're like, “Oh, Yes, this is the driving factor. We should focus all of our work on trying to control its expression”? 

Sydney Pascetta
I don't think we should focus our work on controlling expression. But the one assay in the paper that was called like chromatin immunoprecipitation basically showed that some hypoxia responsive elements. They're called HIFs. They directly - they're transcription factors, and they're also translation factors but in this context are transcription factors. They directly bind Y1 and Y5 and their promoter regions showing that like, it is a hypoxic response to upregulate the transcription of neuropeptide Y1 and Y5 in the context of these two cancers that we look at. So yeah, so definitely they are important for playing your role in that environment.

Michael Lim  
So it's a kind of more of like a positive feedback loop where obviously you don't want to have excessively a hypoxic environment within yourselves. But the fact that because of hypoxia, you get behind that hypoxia inducible factors binding to your receptors and causing stronger effects, some more and more migration more and more movement and possibly causing more cancerous cells and hypoxia ones to develop. Is there a way to stop that feedback loop?

Sydney Pascetta
So, that's what we're trying to understand with our follow up paper. We are currently working on a follow up paper right now. And so, our follow up paper is trying to understand if we can down regulate these biophysiological responses, like the migration, the proliferation, all that kind of stuff. If we can down regulate it by antagonizing the receptors. So in this paper, we're basically stimulating the receptors and we're stimulating them both individually and as a whole. Cause there was the NPY general agonist and there was the Y1 and Y5 specific agonists so we were like, does hypoxia drive the production of Y1 and Y5 in breast cancer? The answer is yes. How is that happening? Well, it might be through the HIFs directly binding and increasing their transcription, and then can hypoxia and the receptor specific agonists combine together to further drive proliferation, migration, all those kinds of things. And the answer that was yes. So, in our follow up paper, we're trying to understand if you lock those receptors, can you antagonize those responses in any way or maybe slow them down?

Michael Lim
So, it's helping your treatments is what you are saying? [Sydney: Yeah]In general, I think cancer research can be relatively difficult to complete and kind of sell the importance of working on specific say, cell lines or working with individuals, even like we just discussed earlier, the importance of how you're going to hold the cell lines terms of temperature and dissolved oxygen levels. So, what is one thing you wish listeners knew, or reconsider how they think about cancer research?

Sydney Pascetta
Honestly, I think the most important thing for people to understand is just how expensive… in general, like research in the molecular sciences is extremely expensive. All the reagents you have to use, all the personnel that we need to use and all that kind of stuff. Our lab, for example, is a really well funded lab and we're like a medium to small sized lab and we go through tens of thousands of dollars of things a month and that's a normal regular month. And so I obviously am a big proponent of adding money to like science research and funding and all that kind of stuff. But the funding climate in general and science is actually pretty poor. I think less than 10% of grants, like people that applied to grants get awarded them and the government doesn't really put it as a top priority to fund science and stuff like that. So it's funny because we're actually very small scale here, but what we do every day is quite expensive. So, scale that up to big corporations and stuff like that. And obviously there's a reason for privatization and stuff. So yeah, so I just think it's important to recognize that the more money that goes into funding these projects, the more forward movements will be made in our understanding of just the world in general, you know.

Michael Lim
I'm not sure how often you read that research in the news, but often you read through the comments like how could it be spending so much money millions of dollars on this research? Well, it's because research just costs a lot of money. Companies know that. And you know, you have no option but to buy from them for them to suddenly jack up the price – What can you do? Research is inherently expensive.

Sydney Pascetta
It's true. It's true and mostly qualified personnel are very underpaid. Postdocs, research associates are very underpaid as well as grad students. So I'm just a really big proponent of like, understanding like where the money is going because, yeah, people are always like “Oh, we raised like a million dollars for like a breast cancer walk for research or something like that. That my fund a medium lab for like, maybe a year. You know, and when you put it into that kind of context, it sounds like so much money, but then we break it down, it's like it goes very fast. So, I think funding the sciences is just like super, super important. All areas of the sciences, not just like biology or molecular biology or anything like that. But all areas of science deserve - deserve more funding, I think.

Michael Lim
So of course, I'd like to ask all my guests because obviously, things don't always go as planned. So, if you go back in time and change one thing about your study, what would it be and why?

Sydney Pascetta 
I don't know because we took a long time to publish this,like I've been working on it for like that I'm entering my fifth year now. It was started before I came to the lab by a previous postdoc and we did six months of revisions. So, I feel like there was like a lot of thought put into the project. It came off the backs of that postdocs, previous postdoc. So yeah, I don't know if I haven't really changed much, where I just feel like we're exploring and trying to understand what's going on more so than having like an end goal or a timeline. So, the follow up paper is more exploration of understanding how these things are working. 

Michael Lim
So that's it for me. There's going to be a couple questions I've taken from social media. And our very first question is, at what age should I be more wary of getting breast cancer? Does signalling activity change with age to become more abnormal, for example, and contribute to becoming a more breast cancer risk?

Sydney Pascetta
Yeah, so it's really does change of age and that's why cancer is often associated with its as a disease of aging. There's a much higher incidence amongst those who are elderly or aging, in general, and the reason for that is just because those normal biological processes that are happening at all times in our in our cells are hijacked, are not working functionally, are working abnormally and that's why cancer develops in the first place. So, we have cancer cells in our bodies at all times. Our immune system is just really good at recognizing them and getting rid of them. But as you age, your immune system becomes more preoccupied or maybe less robust than it used to be because you have a lot of other things going on already and some of those cells kind of like escaped notice sometimes and then they can develop into a mass of cells and then all of those cells kind of develop into a tumor and etc. So, yeah, definitely cancer is a disease of aging. It's also sometimes just, there are genetic associations as well. Like whether your one protein is maybe prone to mutations that would make it maybe nonfunctional or hyperfunctional, or whatever. But I don't think there's like a specific age I think, you know, lifestyle factors also play a role. So just like maintaining a healthy lifestyle, and there's a lot of research that also shows your stress levels and mental health also play a role in the development of disease states and stuff like that. So, I think just generally leading a healthier lifestyle and stuff like that is definitely beneficial.

Michael Lim
I should have really thought about it before that probably everyone has cancerous cells in the body. Like obviously, you know, just the sheer number of cells in your body some of them will be cancerous. But it's a little sobering.

Sydney Pascetta 
Yes it's true, a lot of people maybe don't realize it, you know, there could be like, whatever unforeseen circumstances that maybe your immune system just doesn't protect whatever's going on in that exact moment, but take your vitamins, your multivitamin, exercise [crosstalk].

Michael Lim
So the next question is, are there other symptoms of breast cancer I should be wary of other than a lump? Do you think they will be easier to measure neuropeptide Y and receptor levels in the future as a symptom of breast cancer?

Sydney Pascetta  
So actually, currently the neuropeptide Y family is used as an imaging tool. So yeah, so they basically attach molecules that could be imaged and then kind of send them through and then see how much you have in your breast cancer. Usually, you're diagnosed with breast cancer at that time. But yeah, it is used as a diagnostic tool currently. I don't know if like sheer levels would maybe ever be used as a pure diagnostic tool, but they are definitely used in imaging and stuff like that.

Michael Lim 
I guess the problem that like you mentioned before, it can vary between individuals, your lifestyle unless you have a really good baseline, you're like measuring it every year, your entire life leading up to one day you have a really high spike.

Sydney Pascetta
Yeah, exactly. And as part of our follow up if we do take resections from cancer patients and normal patients as well. And kind of compare them and they do tend to have more higher expression of Y1 and Y5, but that's TBD in the second paper. 

Michael Lim
Your holding secrets back from us. And so, our last question we have, do you think your work will replace other forms of cancer treatments like chemotherapy? How long do you think it'd be before it's more widely used or talked about?

Sydney Pascetta
I mean, I think the hope would be that we don't have to use something as aggressive as like chemo and stuff like that, because that's just basically kills all the cells in your body in the hopes that you irradiate like what whatever is you know, the cancerous hub, right? But the problem with cancer of course, if cancer if the problem with cancer, were just a tumor, we would be able to be able to surgically remove it and every patient walk free and be fine, right? It's those single cells that kind of migrate away from the tumor microenvironment and then establish themselves in other tissues. So that word like metastasis is kind of like a huge negative buzzword, I suppose you could say for cancer. So, if we can, like, understand how those cells in particular are functioning, and maybe target those a bit more effectively, that would be really promising for research in general, cancer research in general. I think we would hope to just generally move away from things that would, you know, make people otherwise very sick and frail and stuff like that.

Michael Lim
Alright, so before we end, if there's someone listening who say is maybe in undergrad or high school and is thinking about what they want to do, do you recommend they also go into cancer research or what words of encouragement or advice did you have for those people?

Sydney Pascetta
I think as scientists, we're all just like curious. I mean, I know for myself, like most of my friends here as grad students don't necessarily care about the specific thing that they research. I mean, some do, but I would say for the most part most of us don’t. I think we just enjoy the curiosity of science and you know, for one moment in time, you're the only person in the world that knows that result or knows that thing. [crosstalk]

Michael Lim
You’re the foremost expert for, you know, however long it takes. [crosstalk]

Sydney Pascetta
But like even like as you're reading an assay or something like that, or waiting for results to come out. You are the only person in the world that knows what those results are and what they might mean. So, I feel like that's the really exciting intangible experience of science. So yeah, like I don't I don't think most of us really, I don't want to say we don't care. That's not the word I'm trying to use. But like, we're just like, [crosstalk] Yeah, we're just getting started to understand things about the world and it does really matter what those things are. 

Michael Lim
And with that we’ve come to the end of today’s podcast. A big thanks again to our guest Sydney Pascetta for joining us today. GryphonCAST is brought to you by your host me Michael Lim with editing assistance from Ian Smith. If you're hoping to learn more about different science topics, please check out SCRIBE Research Highlights that's S-C-R-I-B-E, Scribe Research Highlights on the University of Guelph website at uoguelph.ca. Or you can follow us on social media at UofGCBS. You can find us on Instagram, Twitter, and Facebook. Music in the podcast comes from uppbeat.io they’ll be details in the show notes as always. Until next time, stay curious.